The amino acid sequence of a protein is usually encoded directly by the sequence of nucleotides in a gene. In some cases, however, the correct protein sequence does not correspond exactly to the sequence encoded in the DNA. Instead, the DNA sequence must be edited to produce the functional protein. The editing of sequence information frequently occurs at the level of the messenger RNA (mRNA).
An activity characteristic of an adenosine deaminase enzyme specific for double-stranded RNA (dsRNA) has been previously functionally detected in many different invertebrates and vertebrates [Bass and Weintraub, Cell, 55:1089 (1988); Wagner et al, Proc. Natl. Acad. Sci., USA, 86.:2647 (1989); Rebagliati and Melton, Cell, 48:599 (1987); Bass and Weintraub, Cell, 8:607 (1987); Seiky and Iatrou, J. Mol. Biol., 218:517 (1991)] and also in most mammalian tissues and cell lines [Wagner and Nishikura, Mol. Cell Biol., 8:770 (1988); Wagner et al., Mol. Cell Biol., 10:5586 (1990)]. This enzymatic activity converts multiple adenosine (A) residues to inosines (I) by hydrolyric deamination [A. G. Polson et al, Biochem., 30:11507 (1991)]in both inter- and intra-molecular dsRNAs [Nishikura et al, EMBO J., 10:3523 (1991)], creating I-U mismatched base pairs in dsRNAs. The accumulation of extensive mismatched I-U base pairs in the dsRNA causes unwinding of the RNA double helix.
DRADA requires a double-helix structure for its substrate recognition but lacks strict sequence specificity [Bass and Weintraub, (1988); Nishikura et al., (1991); Wagner et al., (1989), all cited above].
DRADA is the first and so far, the only, RNA-unwinding activity that results in an accompanying base modification on the substrate RNA. This dsRNA unwinding/modifying activity further differs from other dsRNA unwinding activities or RNA helicases in that it seems to bind specifically to dsRNA.
DRADA is anticipated to be responsible for RNA editing of glutamate-gated ion-channel gene transcripts in mammalian brains. The A to I changes introduced in mRNA by this enzymatic activity, called DRADA, are revealed by A to G changes in its cDNA.
Several examples of in vivo interaction of this enzymatic activity with cellular as well as viral gene transcripts have been reported. For instance, maternal fibroblast growth factor gene and also its antisense transcripts seem to be extensively modified by DRADA in Xenopus oocytes undergoing meiosis [Kimelman and Kirschher, Cell, 59:687 (1989)]. The enzyme is responsible for genesis of defective measles virus with biased hypermutation, which results in lethal human CNS diseases, measles inclusion body encephalitis [Cattaneo et al., Virol., 55:255 (1988); Bass et al., Cell, 56:331 (1989)]. Furthermore, an adenosine located in a short stem structure of HIV TAR was reported to be modified to inosine by DRADA in a tat dependent manner [Sharmeen et al., Proc. Natl. Acad. Sci., USA, 88:8096 (1991)].
Because the enzyme introduces changes in the sequence of its substrate RNA, DRADA is anticipated to be involved in the RNA editing process [see, Kim and Nishikura, in RNA Editing, R. Benne, Ed. (Simon and Schuster International, Chichester, England (1993), pp. 179-192]. Three cases of RNA editing of glutamate-gated ion channel subunits which are responsible for the fast excitation of neurons in mammalian brain that always result in conversion of an A in the gene to a G in the cDNA have been reported [Sommer et al., Cell, 67:11 (1991); Verdoorn et al., Science, 252:1715 (1991); Kohler et al., Neuron, 10:491 (1993)]. This RNA editing process replaces the gene encoding glutamine (CAG) with an arginine (CGG in subtype GluR-B,-5, -6), isoleucine (AUU) with valine (GUU), or tyrosine (UAC) with cysteine (UGC in subtype GluR-6) within the transmembrane domains. These amino acid conversions have a large functional significance since the presence of the amino acid residue introduced by RNA editing in the transmembrane domain has been shown to be a determining factor in the channel behavior.
DRADA is thus implicated in conditions or disorders characterized by the malfunction or deficient functioning of neuronal transmission in mammalian brain, e.g., in disorders such as stroke, Huntingdon's disease, Alzheimers disease and other such neurological conditions.
There is a need in the art for the isolation and recombinant production of the protein which produces the enzymatic activity described for DRADA, to enable its use in genetic engineering, recombinant production of useful proteins and drug development and screening.